Genotype‐dependent contribution of CBF transcription factors to long‐term acclimation to high light and cool temperature

Abstract When grown under cool temperature, winter annuals upregulate photosynthetic capacity as well as freezing tolerance. Here, the role of three cold‐induced C‐repeat‐binding factor (CBF1–3) transcription factors in photosynthetic upregulation and freezing tolerance was examined in two Arabidopsis thaliana ecotypes originating from Italy (IT) or Sweden (SW), and their corresponding CBF1–3‐deficient mutant lines it:cbf123 and sw:cbf123. Photosynthetic, morphological and freezing‐tolerance phenotypes, as well as gene expression profiles, were characterized in plants grown from the seedling stage under different combinations of light level and temperature. Under high light and cool (HLC) growth temperature, a greater role of CBF1–3 in IT versus SW was evident from both phenotypic and transcriptomic data, especially with respect to photosynthetic upregulation and freezing tolerance of whole plants. Overall, features of SW were consistent with a different approach to HLC acclimation than seen in IT, and an ability of SW to reach the new homeostasis through the involvement of transcriptional controls other than CBF1–3. These results provide tools and direction for further mechanistic analysis of the transcriptional control of approaches to cold acclimation suitable for either persistence through brief cold spells or for maximisation of productivity in environments with continuous low temperatures.

Leaves of winter annuals grown in cool versus warm temperatures are also thicker and contain more chloroplast-rich mesophyll cells per unit area (Adams et al., 2016;Cohu et al., 2014;Gorsuch et al., 2010).
By virtue of upregulation of photosynthetic capacity in leaves that develop under cool temperatures (Cohu, Muller, Stewart et al., 2013), plants are able to maintain sugar production and transport for underground storage while limiting aboveground growth and exposure of leaves to freezing temperature (Eremina et al., 2016). This enhancement of photosynthesis-related traits illustrates how acclimatory adjustment leads to new homeostasis that minimizes internal stress despite a challenging environment (Anderson et al., 1995).
Notably, a similar upregulation of photosynthesis-related features takes place during acclimation to high growth-light intensity (Boardman, 1977;Gauhl, 1976;Munekage et al., 2015) in many species, including Arabidopsis thaliana (Hoshino et al., 2019;Stewart, Polutchko, Adams, Cohu, et al., 2017;. Common regulatory networks may thus be involved in both cold and high light acclimation, such as signalling networks that respond to the level of excitation pressure in the chloroplast (Anderson et al., 1995;N. Hüner et al., 2012;N. P. A. Hüner et al., 2016).
The transcription factor family of C-repeat-binding factors (CBFs) has been proposed as a regulatory network that may orchestrate photosynthetic upregulation and enhance freezing tolerance in response to growth under cool temperatures and/or high light intensities (N. P. A. Hüner et al., 2016;Savitch et al., 2005). A. thaliana contains three tandemly duplicated CBF paralogs (CBF1, CBF2 and CBF3; abbreviated to CBF1-3 in this text) that are strongly induced by cold temperature and orchestrate transcriptional and physiological changes necessary for enhanced freezing tolerance (Knight & Knight, 2012;Shi et al., 2018;Thomashow, 1999). Laboratory studies revealed largely overlapping functions for the CBF1-3 transcription factors as well as a requirement for combined loss-of-function mutations in all three genes to strongly reduce the induction of freezingtolerance genes and freezing tolerance itself (Gilmour et al., 2004;Jia et al., 2016;Zhao et al., 2016). CBF overexpressing lines exhibited higher freezing tolerance as well as greater leaf thickness, chlorophyll levels and photosynthetic rates per unit area even when grown under low light and warm temperature (Gilmour et al., 2004;Savitch et al., 2005). Thus, CBF overexpression induced both the survival trait of enhanced freezing tolerance and the productivity-maintenance trait of photosynthetic upregulation.
Following a 5-year, reciprocal transplant investigation of two A.
While possessing a similar constitutive freezing tolerance, in warmgrown plants, SW also induced greater freezing tolerance relative to IT when grown under controlled cold conditions (Gehan et al., 2015;Park et al., 2018;Sanderson et al., 2020). Under field growth conditions, the CBF1-3 region was identified as a QTL for fitness (Ågren et al., 2013) as well as freezing tolerance (Oakley et al., 2014). In fact, IT possesses a naturally occurring 8-bp deletion in its CBF2 gene that renders the CBF2 transcription factor nonfunctional (Gehan et al., 2015). Nevertheless, CBF2-deficient lines of SW still maintained greater cold-induced freezing tolerance than IT (Park et al., 2018;Sanderson et al., 2020). Likewise, a CBF1-3-deficient line created in SW maintained greater cold-induced freezing tolerance than a CBF1-3-deficient line created in IT (Park et al., 2018).
In the present study, IT and SW were grown under a factorial design of different light intensity and temperature regimes.
Transcriptome data from fully expanded leaves were generated to compare expression patterns of genes associated with the functional traits of freezing tolerance and photosynthesis and chloroplast redox state (reduction state of the primary electron acceptor of photosystem II [PSII], Q A ) was assessed to address the relationship between chloroplast excitation pressure and CBF1-3 expression levels.
Under the two most different growth conditions, the wild-type ecotypes, IT and SW, were grown alongside the corresponding CBF1-3-deficient mutant lines it:cbf123 and sw:cbf123 (Park et al., 2018). Fully expanded leaves of these plants that had developed under the respective growth conditions were assayed for freezing tolerance, morphological and photosynthetic characteristics and expression of genes associated with the latter phenotypic traits. (Controlled Environments Ltd.) and then in E36-HID (Percival Scientific) growth chambers alongside the corresponding CBF1-3deficient lines it:cbf123 and sw:cbf123 that had been generated by Park et al. (2018) via CRISPR/Cas9 (for more information on the ecotypes, see Adams et al., 2016;Ågren & Schemske, 2012). For selected experiments, sw:cbf2, a CBF2-deficient line created in SW by the same group (Park et al., 2018), was included as well. CBF1-3 genotypes of it:cbf123, sw:cbf123 and sw:cbf2 plants used in this study were confirmed by Sanger sequencing.  (Cohu, Muller, Demmig-Adams et al., 2013, Cohu, Muller, Stewart, et al., 2013. The setpoints for the growth chambers were 25°C/20°C (light/dark) for LLW, 16°C/12.5°C (light/dark) for LLC, 20°C/20°C (light/ dark) for HLW and 8°C/12.5°C (light/dark) for HLC. The controlled conditions chosen here are an approximation of total daily photon input in natural settings. While peak natural light intensity would be higher in exposed sites, the HL condition of 1000 µmol photons m −2 s −1 of continuous light for the duration of the light period approximates total light supply on a clear day at the point of origin for both ecotypes (Adams et al., 2016). The LL growth regime resembles peak light intensities in a shaded environment. All plants were grown from seeds soaked in water at 4°C for 4 days and then germinated in six-pack seed-starting trays containing 50 ml of Fafard Growing Mix 2 (Sun Gro Horticulture) under 9-h photoperiods of either 100 (LLW and LLC) or 1000 (HLW and HLC) µmol photons m −2 s −1 and a common air temperature of 25°C during the photoperiod and 20°C during the dark period. Following germination, individual seedlings were transplanted with 50-ml soil from their respective cells into larger (2.9 L) pots, and then transitioned to their final growth conditions (for details, see Figure S1; see also Cohu, Muller, Stewart et al., 2013). Plants received water daily with nutrients added every other day as previously described . Sampled plants were all nonflowering and of similar size (for plant age, see Figure S1). Unless otherwise specified, samples were taken at the end of the 15-h dark period from young leaves that were no less than two-thirds expanded.

| Leaf phenotypic traits
Leaf photosynthetic capacity was determined as light-and CO 2 -saturated oxygen evolution with leaf disc oxygen electrodes (Hansatech Instruments Ltd.; Delieu & Walker, 1981) as previously described . The reduction state of the primary electron acceptor of PSII, Q A , was assessed via measurements of chlorophyll fluorescence using a pulseamplitude-modulated (PAM) chlorophyll fluorometer (FMS2; Hansatech Instruments Ltd.). Leaves were darkened for 20 min, exposed to a far-red light of 0.6 µmol photons m −2 s −1 for 5 min, and then subjected to 5-min exposures of increasing light intensities. At the end of each 5-min exposure, steady-state fluorescence (Strand et al., 1999) were recorded, maximum fluorescence levels (F m ′) were obtained by applying a saturating pulse of light (0.8 s of 3000 µmol photons m −2 s −1 ) and then minimum fluorescence levels (F o ′) were recorded by briefly darkening the leaf. Q A reduction state was calculated as were conducted in the laboratory at ambient temperature (approximately 22°C) and measurements on HLC plants were conducted inside the growth chamber in which they were grown (with an air temperature of 8°C). Chlorophyll a and b content was determined from leaf discs via high-performance liquid chromatography as previously described (Stewart et al., 2015) or via spectrophotometry as previously described (Arnon, 1949).
Leaf dry mass was measured with an A-160 balance (Denver Instruments Company) from leaf discs that were dried at 70°C for 7 days. For leaf-thickness measurements, leaves were embedded in 7% (w/v) agarose and sectioned into 80-100 µm thick sections using a 752/M Vibroslice tissue cutter (Campden Instruments Ltd.).
Sections were stained with 0.02% toluidine blue O for 30 s, and images were taken approximately 150 μm away from the mid-vein (where no major veins or trichomes were present) with an AxioImager (Zeiss) coupled with a MicroPublisher color camera (QImaging). Leaf thickness was quantified for 10 representative sections of each plant (i.e., 10 technical replicates for each biological replicate) using ImageJ (Schindelin et al., 2012).

| Freezing tolerance assays
Freezing tolerance of leaf tissue was determined via electrolyte leakage assays based on those described by Thalhammer et al. (2014).
Leaves (grown under LLW or HLC conditions) with fresh-cut petioles were placed in 300 ml of deionized H 2 O (petioles submerged) and subjected to subfreezing temperatures using an Arctic A25 refrigerated water bath (Thermo Fisher Scientific) and a cooling rate of 4°C h −1 . Electrical conductivity was measured using an Exstik II probe (Extech Instruments). The data for each replicate were fitted to a four-parameter logistic model and lethal freezing temperatures (LT 50 ) values were determined as the inflection points from these models.
Maximal intrinsic PSII efficiency in darkness was assessed in parallel with the electrolyte leakage assays after overnight incubation on ice (4°C) to thaw frozen leaves for measurements of chlorophyll fluorescence with an Imaging-PAM Maxi (Walz). Minimal fluorescence levels (F o ) were recorded after a 20-min dark period at room temperature following the freezing treatments, and then maximal fluorescence levels (F m ) were recorded by applying a pulse of saturating light (2500 µmol photons m −2 s −1 ). Maximal intrinsic PSII efficiency was calculated as F v /F m = (F m − F o )/F m , and false-coloured images of F v /F m were generated using ImageJ (Schneider et al., 2012).
Freezing tolerance of whole plants was determined via survival assays based on previously described protocols (Sanderson et al., 2020;Xin & Browse, 1998). Seeds were germinated and transferred to LLW or HLC growth conditions as described above with the exception that seedlings were not transferred to individual pots and were instead thinned to prevent overcrowding. After 10 days under LLW or HLC growth conditions, plants with six to eight leaves were transferred to ½ MS-agar plates, chilled to −1°C in the presence of ice chips for 8 h, and frozen overnight (16 h) at an average freezer temperature of −10°C.
To minimize positional variation in temperature in the freezer, sealed plates were put in a tray with ice before placing them into the freezer.
Furthermore, the location of the three replicate plates for each genotype/growth environment pairing was randomized within the ice tray.
Plates were then transferred to 4°C for 1 day, and plant survival was assessed after another 2 days of recovery in LLW conditions. Surviving plants remained green and erect, whereas nonsurviving plants were white and no longer erect.
2.4 | Gene expression analysis of CBF1-3-regulated genes using real-time qPCR RNA extraction, cDNA synthesis and qPCR were performed as previously described (Wakao et al., 2014). All primer pairs were confirmed as having 90%-105% amplification efficiency and linear amplification within their dynamic range in experimental samples using serial dilutions of cDNA before experiments. Relative transcript levels were calculated by the ΔΔC t method (Livak & Schmittgen, 2001) using PEX4 (AT5G25760) as the internal reference. PEX4, a peroxisomal ubiquitinconjugating enzyme, is an established RT-qPCR internal reference (Dekkers et al., 2011) and was confirmed in the RNAseq data set to have constant expression levels in all conditions and ecotypes. Primers were designed using Primer3 (Untergasser et al., 2012) against the 3ʹ-UTR of each gene to avoid binding to off-target paralogous genes. A single peak in melt-curve analysis with a unique melting temperature was observed for each amplicon, verifying that off-target amplification of paralogous genes was negligible.

| RNAseq library preparation and analysis
Two flash-frozen leaf discs of 0.73 cm 2 were homogenized in liquid nitrogen by bead beating, and RNA was extracted and DNase-treated using the Qiagen RNeasy Plant Mini Kit (Qiagen). Integrity of purified RNA was validated using a 2100 Bioanalyzer (Agilent Technologies) and concentration determined using a QuBit fluorometer (Thermo Fisher Scientific). Plant rRNA was depleted from 2 mg of purified RNA using the RiboZero rRNA removal kit for plants (Illumina).
Barcoded cDNA libraries were generated from our rRNA-depleted RNA samples using the TruSeq RNA library preparation kit (Illumina).
Sequencing of barcoded cDNA libraries was performed at the Vincent J. Coates Genomics Sequencing Laboratory using a HiSeq 2500 platform with 50 bp single-end reads (Illumina).

| Statistical analyses
For phenotypic data, comparisons of two means were evaluated via Student's t tests and comparisons of multiple means evaluated via one-way analysis of variance (ANOVA) coupled with post hoc Tukey-Kramer honestly significant differences (HSD) tests. The effects of genotype (e.g., CBF1-3 deficiency) and growth conditions as well as genotype response to the growth conditions for the IT (IT & it:cbf123) and SW (SW & sw:cbf123) genetic backgrounds were each assessed via two-way ANOVA. Nonlinear curves were generated using three-parameter exponential and four-parameter logistic models. All statistical analyses, excluding those of RNAseq data, were conducted using JMP software (Pro 15.0.0; SAS Institute Inc.).
RNAseq statistical analysis was performed using the genomic analysis tools available through Galaxy (Afgan et al., 2018). Quality of RNAseq runs was validated by FastQC and adapter sequences were clipped using FASTQ (Gordon & Hannon, 2017). Reads were mapped to the A. thaliana reference genome (TAIR10) and preliminary differential expression analysis was conducted using HISAT and StringTie (Pertea et al., 2015). Differential expression analysis was conducted using DESeq. 2 as well as the calculation of adjusted p-values, which limit high false positive discovery rates due to multiple testing (Love et al., 2014). Data can be accessed on the Gene Expression Omnibus at GSE154349. Log 2 fold-changes were transformed with the regularized log function to minimize variance caused by low expression genes, then clustered and plotted using pheatmap. In pheatmap, each sample was clustered on the horizontal axis based on the similarity of its transcriptome to the 23 other transcriptomes. On the vertical axis, individual genes were clustered based on the similarity of their expression profile across the 24 samples to the expression profile of other genes. Gene Ontology (GO) enrichment analysis was performed using PANTHER (http://pantherdb.org/) and removal of redundant GO terms was performed using REVIGO (revigo.irb.hr). Before submission to REVIGO, GO-terms with enrichment-values below threefold were eliminated to remove weakly enriched GO terms. In addition to these shared responses, there were substantial differences between IT and SW in how strongly gene expression responded to HLC (Figure 4c, Tables S10-S16). For instance, cold acclimation genes (GO:0009631; Figure 4c For both ecotypes, genes preferentially induced under HLC were enriched for those that had also been induced by CBF1-3 overexpression in the absence of either high light or cool temperatures (Park et al., 2018) (p-values of 10 −19 and 10 −18 for IT and SW, respectively; Figure 4b, Table S17). Moreover, these genes preferentially induced under HLC were also enriched for genes expressed at lower levels in it:cbf123 and sw:cbf123 following sudden transfer from warm growth conditions to 4°C for 24 h (Park et al., 2018) (p-values of 10 −28 and 10 −38 , for IT and SW, respectively; Table S18). Overall, while CBF1-3 target genes (Jia et al., 2016;Park et al., 2018) were strongly induced in both ecotypes in HLC, these genes tended to be more strongly induced in SW compared to the IT ecotype in this condition. Examples for genes in this previously defined CBF1-3-regulated group that were more strongly induced in SW included the cold acclimation-regulating protein kinase CIPK25  Table 1). Remarkably, CBF1-3 deficiency did not impede upregulation of these traits in HLC in the SW background ( Figure 5a-c, Table 1). Despite the difference in chlorophyll a + b content, IT and it:cbf123 did not differ in chlorophyll a/b under either LLW or HLC (Figure 5d). Similar trends were observed for leaf morphology in that IT and it:cbf123 grown under HLC exhibited significant differences, whereas SW and sw:cbf123 did not (Figures 6 and 7). Specifically, leaves were thinner (Figure 6a-c) and rosettes were larger (had a larger diameter) in it:cbf123 compared to IT (Figure 7a-c) in plants grown in HLC. In contrast, leaf thickness was the same (Figure 6a,d,e) and rosette diameter was similar in HLC-grown plants of SW and sw:cbf123 ( Figure 7a,d,e).

| Freezing tolerance
An initial assessment of leaf freezing tolerance was made using electrolyte leakage and chlorophyll fluorescence, where a sharp increase in leakage and/or decrease in intrinsic PSII indicates freezing damage to membranes (Figure 8). While LLW-grown plants of all genotypes exhibited the same low tolerance to freezing damage by these criteria (Figure 8a), HLC-grown plants were shifted to greater tolerance that was also more pronounced in SW compared to IT and was substantially impaired by CBF1-3 deficiency in both backgrounds ( Figure 8b, Table 2). Figure 8c shows these same data transformed to mean lethal temperature (LT 50 ) upon exposure to stress; onset of significant electrolyte leakage occurred with an LT 50 near −5.6°C for all genotypes grown in LLW but was shifted to lower subfreezing temperatures in leaves grown in HLC compared to LLW to varying degrees depending on genotype. LT 50 of freezing tolerance in sw:cbf123 was 3.5°C warmer than that of SW (Figure 8b,c).
Similarly, LT 50 of it:cbf123 was 3.4°C warmer than that of IT ( Figure 8b,c). This greater electrolyte leakage in sw:cbf123 compared to SW and it:cbf123 compared to IT was accompanied by more pronounced freezing-induced depression of intrinsic PSII efficiency F v /F m (Figure 8d). At the same time, the lesser electrolyte leakage in both it:cbf123 and sw:cbf123 lines grown under HLC compared to LLW indicated contributions from CBF1-3-independent freezingtolerance mechanisms.
F I G U R E 4 (a) Hierarchical clustering of the log 2 expression data (via RNAseq) for 7933 genes with an adjusted p-value below 0.01 in one of the pairwise comparisons for differential expression between ecotypes and growth conditions. The three biological replicates for each growth condition/ecotype set are shown as separate columns. (b) Log 2 expression data (via RNAseq) for IT and SW in HLC relative to LLW for genes previously characterized as regulated by CBF1-3 in Col-0 (Jia et al., 2016;Park et al., 2018). CIPK25 and KIN2 are protein kinases participating in cold acclimation signalling (Ding et al., 2019;Thomashow, 1999). COR47, LTI30 and LTI29 each are cold and drought-induced dehydrin proteins (Puhakainen et al., 2004). SUS1 and GOLS3 are stress-induced sucrose synthase and galactinol synthase enzymes, respectively (Maruyama et al., 2009). COR15B is essential for chloroplast membrane integrity during freezing (Thalhammer et al., 2010). (c, d) Significantly enriched gene ontology (GO) categories with a cutoff of a false discovery rate <0.05 and redundant categories removed by Revigo. GO-term fold enrichment (i.e., how many times more frequently a gene belonging to a given GO category is identified in the set of differentially regulated genes relative to chance) is displayed on the x-axis and the size of the circle is proportional to the number of genes belonging to the GO category (see the legend composed of grey circles). The significantly enriched GO categories were calculated using (c) the genes that were significantly downregulated or upregulated in both ecotypes in HLC and those (d) genes whose expression responded more strongly to HLC in one ecotype relative to the other. The results from excised leaves (Figure 8) were complemented by tests of whole-plant survival after overnight exposure to a temperature of −10°C (Figure 9). Whole-plant survival was extremely low in LLW-grown plants of all genotypes and was generally much enhanced by growth under HLC (Figure 9), consistent with the corresponding electrolyte leakage data obtained with leaves ( Figure 8a,b). However, freezing tolerance remained severely impaired in whole plants even in HLC in it:cbf123 plants (Figure 9). In contrast, there was much less impairment of freezing tolerance in sw:cbf123 compared to SW for HLC-grown whole plants (Figure 9), consistent with the trends in electrolyte leakage levels obtained for leaves (Figure 8b). A uniquely heightened sensitivity of HLC-grown IT CBF1-3 deficient whole plants to freezing damage suggests that protective processes operating at the level of the whole plant require the presence of CBF1-3 in IT but not in SW, as is concluded here for a number of other functions.
Sample size was 120 plants per genotype/growth regime pairing for this experiment. Given that the freezing temperature of −10°C was 3.28°C below the measured LT 50 value for IT CBF1-3-deficient plants grown in HLC, it is possible that a sample size of more than 120 may have been necessary to see a few survivors at this freezing temperature.

| CBF1-3-dependent gene expression
This section focuses on selected genes that exhibited response patterns reminiscent of the trends exhibited by photosynthesis and leaf/ plant morphology ( Figure 10, Table 1) as well as selected genes known to be cold regulated ( Figure 11, Table 2). From among 31 genes that were identified as CBF1-3-target genes in prior work (Park et al., 2018) and showed considerable induction under HLC in IT (Figure 4), nine were selected for validation by RT-qPCR with priority given to genes encoding proteins that can be linked to a role in photosynthetic or leaf-morphological acclimation phenotypes based on either previous studies on these proteins or the presence of a protein domain with an established role in acclimation phenotypes.
Expression level of five of these nine genes ( Figure 10, Table 1) exhibited an impact of CBF1-3 deficiency mirroring that on leaf photosynthetic and morphological traits in the two ecotypes. Specifically, these five genes exhibited a strong reduction in the extent of upregulation under HLC compared to LLW in it:cbf123 compared to IT but no to little difference in sw:cbf123 compared to SW. These genes included cold-and salt-responsive protein transmembrane protein AT5G44565 (Figure 10a), sucrose synthase SUS1 (AT5G20830; Figure 10b), cysteine-rich, defensin-like protein LCR69 (AT2G02100; Figure 10c (Table 2).
While the focus of this study was the effect of complete CBF1-3 deficiency, CBF2 deficiency alone caused a subset of CBF1-3regulated genes to be attenuated in HLC-grown plants in sw:cbf2 relative to SW (Table S19). This was associated with a small decrease in freezing tolerance of whole plants, with survivorship still higher than that of IT plants (Table S19). freezing tolerance (see also Cohu et al., 2014;Cohu, Muller, Stewart et al., 2013;Muller et al., 2014;Sanderson et al., 2020). The combination of mechanisms of photosynthetic upregulation can vary given that this upregulation occurs at multiple scales (chloroplast, cell, whole leaf) that contribute differentially depending on plant genotype and environment (for a review, see Demmig- . These changes can allow overwintering species to achieve full acclimation, defined as new homeostasis where internal stress (with signs of oxidative stress) is minimized or absent.
The pronounced acclimation of plant form and function in SW and IT plants grown in HLC conditions was associated with sweeping changes in gene expression, with approximately 5.2% of total leaf transcriptome upregulated and 4.9% downregulated in HLC relative to growth in low light and warm temperature (LLW). The most strongly enriched and unique GO categories in induced in both ecotypes in HLC included polysaccharide catabolism, cold acclimation, glutamine family amino acid metabolism and organic anion transport, which is consistent with the upregulation of photosynthetic capacity and of freezing tolerance. Continued photosynthetic productivity under cool temperatures in the absence of significant growth generates carbohydrate that can be stored in sink tissues  and also contribute to the accumulation of compatible solutes and freezing point depression (Cao et al., 2007;Reyes-Díaz et al., 2006;Wanner & Junttila, 1999).
Pathways repressed in HLC in both SW and IT included those associated with growth hormones. Reduction of rosette expansion under winter conditions, involving decreased rates of cell elongation T A B L E 1 Results of two-way ANOVAs for the effects of CBF1-3 deficiency (cbf123) and growth conditions as well as the interaction of these effects (cbf123 × GC) on leaf photosynthetic capacity (Figure 5a), leaf dry mass per area (Figure 5b), and chlorophyll a + b levels ( Figure 5c) and expression of associated genes (Figure 10) for the IT (i.e., IT and it:cbf123) and SW (i.e., SW and sw:cbf123) backgrounds

IT background SW background
Trait or gene
LCR69 (AT2G02100) *** *** *** n.s. *** n.s. during leaf development (Hoshino et al., 2019;Yano & Terashima, 2004) helps to minimize foliar freezing damage. Pathways repressed in HLC in both SW and IT included not only those associated with growth hormones (e.g., brassinosteroids) but also with water transport. In fact, vascular tissue is one of the targets of growth hormones (Etchells et al., 2017;Fàbregas et al., 2015) and acclimation to cool temperature is associated with adjustments of vascular anatomy (Adams et al., 2016;Cohu, Muller, Demmig-Adams, et al., 2013;Stewart et al., 2016  obviously, cold acclimation genes were consistently more strongly induced in SW in HLC conditions. Beyond this, another example was the induction of glucosinolate biosynthesis, secondary metabolism and sulphur compound metabolism genes specifically in the IT ecotype in HLC, which could be related different demands for pathogen defense at different latitudes (Roberts & Paul, 2006).

| Stronger enrichment in SW versus IT under HLC
Growth under HLC conditions induced cold-acclimation genes in both ecotypes, but more strongly so in SW relative to IT. This pattern is consistent with the greater freezing tolerance and upregulation of photosynthetic capacity in SW compared to IT (see also Cohu, Muller, Stewart, et al., 2013;Stewart et al., 2016) as well as the lesser excitation pressure in the chloroplast (more oxidized Q A reduction state) of HLCgrown SW compared to IT under experimental high-light exposure. The stronger downregulation of genes involved in light-harvesting in HLCgrown IT suggests that IT limits excitation pressure by lowering lightcollection capacity, which is consistent with the lower Q A reduction state under very low light (when thermal dissipation is not triggered) in HLC-grown IT compared to SW as well as IT's lower chlorophyll a + b content and higher chlorophyll a/b ratio that are indicative of a smaller antenna size (due to preferential degradation of the outer, chlorophyll b-containing light-harvesting complexes). This is consistent with previous studies in which SW increased, rather than decreased, light absorption during cold acclimation and apparently limited excitation pressure by greater utilisation of excitation energy in photosynthetic electron transport (Cohu, Muller, Stewart, et al., 2013), as well as greater photoprotective thermal dissipation (Oakley et al., 2018). Our present findings in HLC growth conditions indicate that the acclimatory adjustments in SW are more conducive to productivity maintenance, while adjustments in IT still mitigate oxidative stress.

| Stronger enrichment in IT compared to SW under HLC
The well-characterized phenotypic features of cold acclimation do occur in IT, but to a lesser extent than in SW. It is noteworthy that the genes more strongly induced under HLC in IT compared to SW have been implicated in abiotic stress responses, as was reported for chloroplast glucose-6 phosphate/phosphate translocator GPT2 (Dyson et al., 2015), chloroplast envelope K + /H + antiporter KEA2 (Kunz et al., 2014), light-harvesting complex LHCB4.3 (Klimmek et al., 2006), cytosolic phosphofructokinase (Kant et al., 2008), cytosolic fumarase (Pracharoenwattana et al., 2010), ferritins (Petit et al., 2001) and pyridoxal phosphate synthase (Denslow et al., 2007).
Future research should further test the hypothesis that both SW and IT make acclimatory adjustments that limit oxidative stress under HLC conditions, but that changes in SW focus more on the enhancement of productivity (which also lowers excitation pressure more effectively), while IT undergoes alternative evasive changes that are somewhat less effective in controlling excitation pressure.
Moreover, genes exhibiting greater downregulation in HLC in IT compared to SW were those involved in growth and signalling (response to the absence of light and hemicellulose metabolism).
T A B L E 2 Results of two-way ANOVAs for the effects of CBF1-3 deficiency (cbf123) and growth conditions as well as the interaction of these effects (cbf123 × GC) on freezing tolerance of discs from fully expanded leaves (LT 50 ; Figure 8) and immature, whole plants of six to eight leaves (% survival; Figure 9) and expression of associated genes from mature leaves (Figure 11) for the IT (i.e., IT and it:cbf123) and SW (i.e., SW and sw:cbf123) backgrounds IT background SW background Trait or gene cbf123 Growth condition cbf123 × GC cbf123 Growth condition cbf123 × GC Freezing tolerance, LT 50 *** *** *** *** *** ** Freezing tolerance, % survival *** *** *** * *** * GolS3 (AT1G09350) *** *** *** *** *** *** CIPK25 (AT5G25110) *** *** *** *** *** *** Previous studies had shown that CBF1-3 are required for full induction of freezing tolerance in mature plants grown under warm conditions and transferred in one step to chilling conditions (Jia et al., 2016;Park et al., 2018;Zhao et al., 2016). However, as was also concluded from studies on warm-grown CBF1-3-deficient mutants abruptly transferred to cold conditions (Jia et al., 2016;Park et al., 2018;Zhao et al., 2016) induction of genes previously defined as CBF1-3-target genes was reduced to varying degrees, but was not fully blocked in CBF1-3deficient lines grown under HLC. The response to sudden exposure to freezing temperature in HLC-grown wild type and CBF1-3deficient lines closely resembled that of plants of the same genotypes after a two-week-long exposure to colder, near-freezing temperatures (air temperature = 4°C in low light, Park et al., 2018). This is noteworthy since the HLC conditions used here involve only cool temperatures (maximum leaf temperature = 16°C, air temperature = 8.5°C) considerably above freezing. This similarity in responses suggests that long-term acclimation to a moderately cool temperature can produce similar results as a shorter-term acclimation to a colder temperature. Furthermore, the presence of high light intensities under the HLC conditions may make a contribution given that long-term acclimation to HLC conditions strongly induced CBF1-3 expression in both ecotypes whereas LLC conditions did not.
The striking difference in the extent to which CBF1-3-deficiency differentially impairs aspects of the acclimation process to HLC conditions in IT compared to SW is a key finding of the present study.
While many genes previously defined as CBF1-3-responsive genes did exhibit strongly reduced expression in both CBF1-3-deficient lines, and may be associated with functions we did not characterize in this study, some genes instead exhibited trends matching those of photosynthetic acclimation and freezing tolerance of whole plants.
For the latter genes, sw:cbf123 compared to SW exhibited little or no difference as the result of CBF1-3-deficiency, whereas it:cbf123 exhibited strongly reduced expression compared to IT. The central features of the acclimation of plant form and function to HLC, that is, photosynthetic upregulation (and its associated morphological traits) as well as freezing tolerance, were only modestly impacted in sw:cbf123 but were strongly impacted (especially in whole plants for the case of freezing tolerance) in it:cbf123 compared to IT. These findings provide further indication for a role of CBF1-3-independent pathways in HLC acclimation of photosynthesis and freezing tolerance and suggest a greater contribution of such pathways in SW.

| Role of paralog compensation
Could a closely related transcription factor be responsible (via paralog compensation) for the induction of CBF1-3-target genes in CBF1-3deficient mutants in HLC? CBF1-3 belong to the ERF/AP2 A-1 subfamily that includes three additional members located outside the CBF1-3 gene locus in A. thaliana (Mizoi et al., 2012). These three other ERF/AP2 A-1 subfamily members (DDF1, AT1G12610; DDF2, AT1G63030; CBF4, AT5G51990) were not expressed at detectable levels in leaf tissue of IT or SW under any of the four growth regimes in either the present study or a previous study (Park et al., 2018). This finding indicates that the latter transcription factors are either not involved or no longer active after long-term acclimation.

| SW as a high-light adapted ecotype
It was previously shown that SW responds with stronger upregulation of photosynthetic capacity and associated leaf features than IT to growth in high light under warm temperature . Based on the latter response, SW was classified as having a high-light phenotype (Adams et al., 2016).
Photosynthetic upregulation is a developmental process involving changes at the organelle, cell, tissue and whole plant levels (Hoshino et al., 2019;Yano & Terashima, 2004), and involves the integration of multiple regulatory pathways, including photoreceptors, photosynthesis-related sugar and redox signals and phytohormone signals. For example, mutants in blue-light photoreceptor signalling and foliar sucrose (Hoshino et al., 2019;Katagiri et al., 2016;Kozuka et al., 2011;López-Juez et al., 2007) have an effect of similar magnitude in increasing leaf thickness in HL-grown plants to those observed for CBF1-3-dependent leaf thickening in the it:cbf123 mutant under HLC. The sucrose synthase SUS1 may contribute to the differential leaf thickening phenotype of SW and IT in HLC conditions via sucrose-responsive leaf thickening (Katagiri et al., 2016) given that (a) it was induced in both ecotypes in HLC conditions, but more strongly in SW, and (b) its induction was unchanged in sw:cbf123 but significantly attenuated in it:cbf123 relative to each respective parental ecotype. Thus, its induction pattern in HLC closely mirrors the trends for leaf thickness reported here. In summary, the present findings suggest that light-responsive signalling pathways with overlapping functions compensate fully for CBF1-3 deficiency in sw:cbf123 with respect to upregulation of photosynthetic capacity and associated leaf features, which were unaffected in sw:cbf123, but significantly (albeit modestly) reduced in it:cbf123. The particularly pronounced photosynthetic upregulation in SW is presumably demanded by the continuously low temperatures at its high-latitude site of origin, whereas the IT ecotype encounters intermittent cold spells (requiring oxidative-stress mitigation) and can quickly resume photosynthetic activity upon return to milder temperatures (for temperature profiles at the respective sites of origin, see Adams et al., 2016).

| CBF1-3 and the nature of acclimation
The more pronounced photosynthetic upregulation in SW plants grown from seedling stage under HLC suggest an acclimation response directed at enhanced productivity in addition to mitigation of oxidative stress. Furthermore, the lesser excitation pressure (lower Q A reduction state) in HLC-grown plants of SW compared to IT represents a lesser trigger for further acclimatory adjustment and evidence of more complete acclimation to HLC conditions in SW compared to IT plants (see also Adams et al., 2013;Cphu et al., 2014;Cohu, Muller, Demmig-Adams, et al., 2013, Cohu, Muller, Stewart, et al., 2013Stewart, Polutchko, Adams, Cohu, et al., 2017).
Additionally, as described above, genes involved in plant response to oxidative stress were consistently more strongly induced in IT CBF ACTIVITY IN HIGH LIGHT AND COOL GROWN ECOTYPES | 407 relative to SW under HLC growth conditions. Rather than maximising excitation energy utilization for photosynthetic energy production and thereby minimising oxidant production, IT thus apparently employs multiple mechanisms that mitigate oxidative stress. CBF1-3 may play a prominent role in the mitigation of oxidative stress in IT, and presumably also during the initial stages of cold acclimation (Fowler & Thomashow, 2002) compared to completed acclimation in SW (see Park et al., 2018). This difference in transcriptional control in SW and IT may stem from evolution under the different environmental conditions at the sites of origin, where IT can presumably 'wait out' infrequent, short-duration cold spells, while it is advantageous for SW to maintain productivity throughout long stretches of cool conditions. These contrasting strategies would be of interest for agriculture in locations with either short cold spells or continuously low temperatures.
In conclusion, several lines of evidence at the transcriptomic and physiological levels are consistent with the CBF1-3-dependent pathway playing a disproportionately greater role under HLC in IT but not in SW. It should be noted that this trend was already evident in young plants and not only in more mature plants. The system of IT and SW, and their CBF1-3-deficient mutants, can serve as a resource to further study CBF1-3-regulated genes that mitigate oxidative stress before, or in the absence of, fully regained productivity as well as genes that remain active after productivity has been fully restored.
In addition, CBF1-3-independent pathways that contribute to full HLC acclimation can also be studied in the SW background. Tools for phenotyping and transcriptional profiling of Recombinant Inbred Line populations are available for these two populations (Ågren et al., 2013;Oakley et al., 2018).

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